Tumor Necrosis Factor (TNF) and interleukin-1 (IL-1) have been associated with a wide range of biological processes, including inflammation. Recruitment of immune cells to sites of injury involves the concerted interactions of a large number of soluble mediators, and several cytokines appear to play key roles in these processes, particularly IL-1 and INF. Both of these cytokines are derived from mononuclear cells and macrophages, along with other cell types. IL-1 and TNF produce many of the same proinflammatory responses, including fever, sleep and anorexia, mobilization and activation of polymorphonuclear leukocytes, induction of cyclooxygenase and lipoxygenase enzymes, increase in adhesion molecule expression, activation of B-cells, T-cells and natural killer cells, and stimulation of production of other cytokines, and TNT also contribute to the tissue degeneration arising from chronic inflammatory conditions, such as stimulation of fibroblast proliferation and induction of collagenase. These cytokines have also been implicated in the process of bone resorption and adipose tissue regulation. Thus, IL-1 and TNF play key roles in a large number of pathological conditions, including rheumatoid arthritis, inflammatory bowel disease, diabetes, obesity, bone mass loss, cancer, neurological conditions such as ischemic stroke or closed head injuries
NF-.kappa.β is a heterodimeric transcription factor regulating the expression of multiple inflammatory genes. The expression of more than 70 known proteins is transcriptionally regulated by the binding of NF-.kappa.β to specific sequence elements in the promoter region of these genes (Baeuerle and Baichwal, Advances in immunology 65:111-137, 1997) NF-.kappa.β has been implicated in many pathophysiologic processes including angiogenesis (Koch et al., Nature 376:517-519, 1995), atherosclerosis (Brand et al., Clint Inv. 97:1715-1722, 1996), endotoxic shock and sepsis (Bohrer et al., J. Clin. Inv. 100: 972-985, 1997), inflammatory bowel disease (panes et al., Am. J. Physiol. 269:H1955-H1964, 1995), ischemia/reperfusion injury (Zwacka et al., Nature Medicine 4: 698-704, 1998), and allergic lung inflammation (Gosset et al., Int Arch Allergy Immunol. 106: 69-77, 1995). Many immune and inflammatory mediators including TNF.α, lipopolysaccharide (LPS), IL-1, anti-CD28, CD40L, FasL, viral infection, and oxidative stress have been shown to lead to NF-.kappa.β activation. Because of the central role of NF-.kappa.β in inflammatory disease, inhibition of NF-.kappa.β by targeting regulatory proteins in the NF-.kappa.β activation pathway represents an attractive strategy for generating anti-inflammatory therapeutics.
The identification and characterization of kinases that phosphorylate I.kappa.βs has led to a better understanding of signaling pathways involving NF-.kappa.β activation. Several different subtypes of IKK have been identified thus far. IKK.α was initially identified as an I.kappa.β kinase induced by TNF.α stimulation in HeLa cells (DiDonato et al., (1997) Nature 388, 548-554). Another I.kappa.β kinase homologous to IKK.α was identified, termed IKK.β and determined to be the major I.kappa.β kinase induced following TNF.α stimulation (Takeda et al., (1999) Science 284, 313-316; Hu et al., (1999) Science 284, 316-320; Li et al., (1999) Science 284, 321-325; Pot et al., (2000) U.S. Pat. No. 6,030,834; Woronicz & Goeddel (1999) U.S. Pat. No. 5,939,302). IKK.α and IKK.β have an overall homology of 52% and a 65% homology in the kinase domain (Zandi et al., (1997) Cell 91, 243-252).
I.kappa.β protein kinases (IKKs) phosphorylate I.kappa.βs at specific serine residues. For example, they specifically phosphorylate serines 32 and 36 of I.kappa.β.α (Traenckner et al., (1995) EMBO J. 14, 2876-2883; DiDonato et al., (1996) Mol. Cell. Biol. 16, 1295-1304). Phosphorylation of both sites is required to efficiently target I.kappa.βa for degradation. Furthermore, activation of IKK.α and IKK.β is usually in response to NF-.kappa.β activating agents and mutant IKK.α and IKK.β, which are catalytically inactive, can be used to block NF-.kappa.β stimulation by cytokines such as INF.α and IL-1 (Rgnier et al., (1997) Cell 90, 373-383; Delhase et al., (1999) Science 284, 309-313). I.kappa.β protein kinases are therefore essential in the regulation of NF-.kappa.β activation processes.
IKK.α and IKK.β have distinct structural motifs including an amino terminal serine-threonine kinase domain separated from a carboxyl proximal helix-loop-helix (H-L-H) domain by a leucine zipper domain. These structural characteristics are unlike other kinases, and the non-catalytic domains are thought to be involved in protein-protein interactions. Proteins which bind to IKKs may therefore be capable of regulating the activity of NF-.kappa.β (Marcu et al., (1999) U.S. Pat. No. 5,972,655) and potentially regulating downstream events such as induction of NF-.kappa.β.
Inflammation is defined as the reaction of vascutarized living tissue to injury. As such, inflammation is a fundamental, stereotyped complex of cytologic and chemical reactions of affected blood vessels and adjacent tissues in response to an injury or abnormal stimulation caused by a physical, chemical or biological agent. Inflammation usually leads to the accumulation of fluid and blood cells at the site of injury, and is usually a healing process. However, inflammation sometimes causes harm, usually through a dysfunction of the normal progress of inflammation. Inflammatory diseases are those pertaining to, characterized by, causing, resulting from, or becoming affected by inflammation. Examples of inflammatory diseases or disorders include, without limitation, asthma, lung inflammation, chronic granulomatous diseases such as tuberculosis, leprosy, sarcoidosis, and silicosis, nephritis, amyloidosis, rheumatoid arthritis, ankylosing spondylitis, chronic bronchitis, scleroderma, polymyositis, appendicitis, inflammatory bowel disease, ulcers, Sjorgen's syndrome, Reiter's syndrome, psoriasis, pelvic inflammatory disease, orbital inflammatory disease, thrombotic disease, and inappropriate allergic responses to environmental stimuli such as poison ivy, pollen, insect stings and certain foods, including atopic dermatitis and contact dermatitis.
Inflammatory diseases present a worldwide problem. Studies of disease burden have re-affirmed that tuberculosis is among the top 10 causes of death in the world, Asthma affects 5% of the adult population and 10-15% of the population of children (Armetti and Nicosia (1999) Boll Chim. Farm. 138(11): 599). Asthma is a chronic inflammatory disease that is associated with widespread but variable airflow obstruction.
Sepsis is yet another inflammation disorder and is caused by the presence of various pus-forming and other pathogenic microbes, or their toxins, in the blood or tissues of a subject. Sepsis is characterized by a systemic inflammatory response to bacterial products during infection. The symptoms of sepsis, such as fever, are caused at least in part by the inflammatory response of the body to the infecting agent.
Because of the important role played by TNF and IL-1 in many pathological conditions, and the involvement of IKK.α and IKK.β in the signal transduction of both TNF and IL-1, there is a need for compounds that potently and selectively inhibit either of these IKK kinases, as well as treatments or therapies using such compounds. The present invention satisfies these needs.
Recently, double-stranded RNA molecules (dsRNA) have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi). WO 99/32619 (Fire et al.) discloses the use of a dsRNA of at least 25 nucleotides in length to inhibit the expression of the IKK-B gene in C. elegans. dsRNA has also been shown to degrade target RNA in other organisms, including plants (see, e.g., WO 99/53050, Waterhouse et al.; and WO 99/61631, Eleifetz et al.), Drosophila (see, e.g., Yang, D., et al., Curr. Biol. (2000) 10:1191-1200), and mammals (see WO 00/4.4895, Limmer; and DE 101 00 586.5, Kreutzer et al.). This natural mechanism has now become the focus for the development of a new class of pharmaceutical agents for treating disorders that are caused by the aberrant or unwanted regulation of a gene.
Despite significant advances in the field of RNAi and advances in the treatment of inflammation, there remains a need for an agent that can selectively and efficiently silence the IKK-B gene using the cell's own RNAi machinery that has both high biological activity and in vivo stability, and that can effectively inhibit expression of a target IKK-B gene for use in treating inflammation.